This proposal is designed to unify the biochemical and genetic aspects of NAD metabolism using the enteric pathogen Salmonella typhimurium. Mutants deficient in several pyridine nucleotide cycle enzymes will be isolated on the basis of analog--resistant analog--sensitivity, ethylmethane sulfonate and nitrosoguanidine mutagenesis. Regulatory control mechanisms for both the de novo biosynthetic NAD pathway and the salvage pyridine nucleotide cycles will be analyzed through the isolation of nad-lac and pnc-lac operon fusions. S. typhimurium possesses at least two pyridine nucleotide cycles which serve to recycle intracellular NAD. Regulation of NAD turnover, in conjunction with regulation over de novo biosynthesis, functions in the control and maintainance of intracellular NAD levels. We will attempt to analyze the flow of pyridine nucleotides through the recycling pathways by isolating mutants blocked at the initial steps of NAD degradation, NAD pyrophosphatase and DNA ligase, using Tn10 transposon technology and recombinant DNA techniques. In addition, the only pnc locus thought to be controlled via repression will be cloned and undergo restriction analysis. The significance of the proposed research lies in the intrinsic importance of NAD to overall cellular physiology both as a cofactor and as a substrate. However, little is known regarding the metabolism of this nucleotide or the genetics associated with its regulation. This study will serve to uncover the genetic aspects of a fundamental metabolic pathway.